Long noncoding RNA LINC00152 promotes cell proliferation through competitively binding endogenous miR-125b with MCL-1 by regulating mitochondrial apoptosis pathways in ovarian cancer

Abstract

Recently, an increasing number of studies have focused on the key function of long noncoding RNAs (lncRNAs) in biological activity. Abnormal lncRNA expression was found to relate to the development and pathogenesis of multiple cancers. LncRNA LINC00152 served as an oncogene in multiple cancers; however, its role in ovarian cancer remains unknown. In our research study, LINC00152 was upregulated in ovarian cancer tissues and cell lines. An increasing LINC00152 level was positively correlated with the histological grade, clinical stage, and poor prognosis of ovarian cancer patients. In addition, knockdown of LINC00152 reduced cell growth, induced cell apoptosis, and suppressed tumor growth. Moreover, we revealed that LINC00152 and Myeloid cell leukemia-1 (MCL-1) were targeted by miR-125b and had the same miR-125b combining site. The miR-125b level was negatively correlated with the expression of LINC00152, while MCL-1 was positively related to the LINC00152 level. MiR-125b could affect LINC00152 levels as evaluated by qRT-PCR. Finally, we affirmed that LINC00152 mediated cell proliferation by affecting MCL-1 expression and MCL-1-mediated mitochondrial apoptosis pathways and by working as a competitive endogenous RNA (ceRNA) of miR-125b. In summary, based on ceRNA theory, the combined research on miR-125b and MCL-1, and taking LINC00152 as a new study point, we provide new insight into the molecular mechanism of reversing cell proliferation in ovarian cancer.

Keywords: LINC00152; MCL-1; MiR-125b; cell proliferation; competitive endogenous RNA; long noncoding RNA; mitochondrial apoptosis pathways; ovarian cancer.

Figure 1

LINC00152 is upregulated in ovarian cancer tissues and cell lines. A, qRT‐PCR was performed to detect the expression of LINC00152 in paired normal and ovarian cancer tissues (n = 20). B, ISH was performed to detect LINC00152 expression in ovarian cancer tissues and normal ovarian tissues; magnification, 100× (left), 400× (right). C, Quantification of ISH staining. IOD, integral optical density. D, Survival cure showed that patients with low LINC00152 had a better survival rate than those of with high LINC00152 expression. E, qPCR was performed to detect the expression of LINC00152 in ovarian cancer cell lines (HO‐8910, A2780, and SKOV3) and normal human ovarian epithelial cells (HOSE). Error bars represent the mean ± SD of triplicate experiments, *P < .05, **P < .01, ***P < .001

Figure 2

Downregulation of LINC00152 inhibits the cell proliferation of ovarian cancer cells. A, qRT‐PCR was performed to detect the expression of LINC00152 in SKOV3 and A2780 cells after transfection with sh‐LINC00152. The cells treated with sh‐NC were used as control. B,C, Knockdown of LINC00152 inhibited the proliferation of SKOV3 (B) and A2780 (C) cells detected by CCK8 assay. D, Downregulation of LINC00152 induced an increased apoptosis rate inSKOV3 and A2780 cells. E, Quantification of the apoptosis rate in (D). F, Hoechst 33258 stain assay detected the apoptosis cells in LINC00152Silencingof SKOV3 and A2780 cells. Error bars represent the mean ± SD of triplicate experiments, **P < .01

Figure 3

Identification of miR‐125b as a target of LINC00152. A, Alignment of potential LINC00152 sequences with miR‐125b as identified by miRcode (http://www.mircode.org). B, qRT‐PCR was performed to detect the expression of miR‐125b in paired normal and ovarian cancer tissues (n = 20). C, The correlation between theLINC00152 expression level and miR‐125b level was measured in 20 ovarian cancer tissues (P < .001). D, miR‐125b is downregulated and overexpression in SKOV3 and A2780cells compared to control groups as determined by qRT‐PCR. The expression of miR‐125b was normalized to U6. E, The expression of LINC00152 was upregulated after silencing miR‐125b in SKOV3 and A2780 cells. F,G, The relative luciferase activities were inhibited in the SKOV3 (F) and A2780 (G) cells cotransfected with wild‐type LINC00152 vector and pre‐miR‐125b, and not with the mutant‐type vector. Firefly luciferase activity was normalized to Renilla luciferase. H, Association of LINC00152 and miR‐125b with Ago2 in SKOV3 cells. Cellular lysates from SKOV3 cells were used for RIP with antibody against Ago2. LINC00152 and miR‐125b expression levels were detected using qRT‐PCR. Error bars represent the mean ± SD of triplicate experiments, *P < .05, **P < .01, ***P < .001

Figure 4

MCL‐1 was regulated by LINC00152 through competitive binding miR‐125b. A, Alignment of MCL‐1 sequences with miR‐125b as identified by Microrna (http://www.microrna.org/microrna). B, qRT‐PCR was performed to detect the expression of MCL‐1 in paired normal and ovarian cancer tissues (n = 20). C, The correlation between LINC00152 expression level and MCL‐1 level was measured in 20 ovarian cancer tissues (P < .001). D, The 3′‐UTR of MCL‐1 was fused to the luciferase coding region and cotransfected into SKOV3 and A2780 cells withmiR‐125b mimics to confirm that MCL‐1 is the target of miR‐125b. MCL‐1 3′‐UTR and miR‐125b mimics constructs were cotransfected into SKOV3 and A2780 cells with plasmids expressing LINC00152 or with a control vector, and the relative luciferase activity was determined at 48 h after transfection. E, Western blot analysis validated miR‐125b targets of Mcl‐1 after in SKOV3 and A2780 cells after transfected with sh‐LINC00152, or together with anti‐miR‐125b. β‐actin was used a loading control. F, Quantification analysis of MCL‐1 expression by IPP6.0. Error bars represent the mean + SD of triplicate experiments, *P < .05, **P < .01, ***P < .001

Figure 5

Knockdown of LINC00152 inhibits ovarian tumor growth in vitro. A, CCK8 assay was performed to detect cell proliferation in SKOV3 and A2780 cells after transfection with sh‐LINC00152 under the presence or absence of miR‐125b inhibitor. B, Colony‐forming unit assays. C, Quantification analysis of clone formation efficiency of (B). D, Hoechst 33258 stain assay detected the apoptosis cells. E, Flow apoptosis assay. F, Quantification of the apoptosis rate in (E). Error bars represent the mean ± SD of triplicate experiments, *P < .05, **P < .01, ***P < .001

Figure 6

Knockdown of LINC00152 inhibits ovarian tumor growth in vivo. A, The SKOV3 cells transfected with sh‐LINC00152 alone or together with anti‐miR‐125b were injected into nude mice. The cells transfected with empty plasmid were used as negative control. B, The tumors obtained from mice on day 28 after injection. C, Tumor volumes were calculated every week. D, Representation of MCL‐1 expression in xenografts tumor using the Western blot method in sh‐NC or sh‐LINC00152 with or without anti‐miR‐125b group, respectively. E, Quantification analysis of MCL‐1 expression by IPP6.0. Error bars represent the mean ± SD of triplicate experiments, ***P < .001

Figure 7

LINC00152 modulates the effect of miR‐125b on cell apoptosis protein expression. A, Western blot analysis of apoptosis protein expression in SKOV3 cells transfected with anti‐miR‐125b,sh‐LINC00152or both. B, Quantification analysis of MCL‐1, Bcl‐2, Bax, and cytochrome c expression in (A). C, Quantification analysis of cleaved caspase9, cleaved caspase3, and cleaved PARP level in (A). D, Representation of cleaved caspase3 and cytochrome c expression in xenografts tumor using Western blot method in sh‐NC, sh‐LINC00152 with or without anti‐miR‐125b group, respectively. E, Quantification analysis of cleaved caspase3 and cytochrome c expression. Error bars represent the mean ± SD of triplicate experiments, *P < .05, **P < .01, ***P < .001

Figure 8

LINC00152 oncogenic activity is in part through regulation of mitochondrial apoptosis pathway. A, Western blot analysis of Bax, cleaved caspase3, and cytochrome c expression in SKOV3 cells treated with Bax channel block, sh‐LINC00152, or both. B, Quantification of Bax, cleaved caspase3, and cytochrome c expression in (A). C, CCK8 assay. D, Clone formation assay. E, Quantification analysis of clone formation efficiency of (D). F, Hoechst 33258 stain assay detected apoptotic cells. G, Flow apoptosis assay. H, Quantification of the apoptosis rate in (G). Error bars represent the mean ± SD of triplicate experiments, *P < .05, **P < .01, ***P < .001